UV light treatment to remove cells within a cell culture device

Abstract

Provided herein are methods and devices that use spatial patterning of ultraviolet-C (UV-C) exposure to selectively remove cells from a cell culture device.

Description

UV LIGHT TREATMENT TO REMOVE CELLS WITHIN A CELL CULTURE DEVICECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 604,209, filed November 30, 2023, which is incorporated by reference.BACKGROUND

[0002] The invention is directed to methods and devices for removal of unwanted cells within a cell culture device.

[0003] Exemplary cell culture delivery devices that can be used with the methods herein are described in U.S. Patent Nos.: 8,445,280; 9,446,169; 8,846,307; and 8,003,388, each of which is incorporated herein by reference in its entirety.SUMMARY

[0004] Methods and devices are provided that use spatial patterning of ultraviolet-C (UV-C) exposure to selectively remove cells from a cell culture device. In some embodiments, unwanted cells are removed from regions in a 2D environment while leaving cells within a 3D environment (e.g., a collagen chamber) intact. In some embodiments, methods provide for seeding a cell culture device with mammalian cells, culturing the mammalian cells within the cell culture device, and exposing a preselected region of the cell culture device to UV-C light in an amount and / or intensity and for a period of time effective to kill, inactivate or detach the mammalian cells in the preselected region.

[0005] In some embodiments, the UV-C light includes a laser, a light emitting diode (LED), a light box, a lamp, and a laser. In embodiments UV-C light is transmitted through a microscope objective to selectively remove cells.

[0006] In one aspect, the preselected region can be an injection portion for delivering the mammalian cells to the cell culture device. In other embodiments, the preselected region is a region surrounding a 3D environment of the cell culture device, e.g., a collagen chamber. In some embodiments, the preselected region is a region in the cell culture device where removal of cells is desired.

[0007] In other aspects, the UV-C light is applied at a wavelength between 200-300 nm. In certain exemplary embodiments, the UV-C light can be at a wavelength between 260-280 nm.

[0008] In some embodiments, the preselected region is exposed to the UV-C light for a time period ranging from 1 second to 2 minutes.

[0009] In certain aspects, the UV-C light passes through at least a layer of a polymeric organosilicon compound, silicone, polydimethylsiloxane (PDMS), polystyrene, polycarbonate, quartz, Topas® 8007x10, a cyclic olefin copolymer, or other UV-C transmissive material. In some embodiments, the silicone includes poly dimethylsiloxane (PDMS).

[0010] In other embodiments, the preselected region can be exposed multiple times with the UV- C light.

[0011] In some embodiments, the preselected region is exposed at an interval of every 2 hours, every 6 hours, every 12 hours, every 24 hours, or every 48 hours.

[0012] In other aspects, at least about 70% or more of the mammalian cells are removed from the preselected region.

[0013] In another embodiment, the method can also include washing the killed, inactivated or detached mammalian cells from the cell culture device. For example, the washing includes washing with a cell culture media or buffer.

[0014] In other embodiments, the method can further include flowing a fluid through the cell culture device when exposing the preselected region to UV-C (e.g., flowing a fluid through the cell culture device while simultaneously exposing the preselected region to the UV-C light).

[0015] In some embodiments, the UV-C light is exposed in a spatial pattern to the preselected region. For example, the pattern can include a pulse that removes cells with a UV-C exposure. In embodiments, the UV-C exposure is a small exposure of UV-C light, where a short exposure is 2 seconds, 1.5 seconds or 1 second.

[0016] In some embodiments, the UV-C exposure is a low intensity energy and long exposure. In embodiments, the UV-C exposure is a high intensity energy and a short exposure. In embodiments, a short exposure is 2 seconds, 1.5 seconds, or 1 second. In embodiments, a long exposure is greater than 2 seconds, or 2.5 seconds, or 3 seconds, or 3.5 seconds, or 4 seconds, or 4.5 seconds or 5 seconds or 5.5 seconds or 6 seconds or 6.5 seconds, or 7 seconds or 7.5 seconds, or 8 seconds, or 8.5 seconds, or 9 seconds, or 9.5 seconds, or 10 seconds.

[0017] In some embodiments, the spatial pattern includes an area of as applied to a cell tubule as constructed in the cell culture device. The cell tubule ranges from 5-8 mm long, and 125-500 pm in diameter.

[0018] In embodiments, the spatial patterning is over a wide range. For the basic removal of cells in 2D (cell injection port, channels leading to and from collagen tube), the area includes 1 mm2, or 1.5 mm2, or 2 mm2, or 2.5 mm2, or 3 mm2, or 3.5 mm2, or 4 mm2, or 4.5 mm2, or 5 mm2.

[0019] To pattern cells in the cell tubule itself (removal of select cells from 3D environment), may require high magnification lensing, like that used on an inverted microscope, to expose small areas on the order of 1 OOum x 100 um

[0020] In another aspect, at least a portion of the preselected region can further include a mask that blocks the UV-C light. For example, the mask can include a metal or a non-UV-C transmissive material.

[0021] In some embodiments, the cell culture device is made of a UV-C transmissive material. In embodiments, transmission is the amount of light that passes through a sample as compared to the amount of light that passes through a control. In embodiments transmission is expressed in percent. In embodiments, all materials UV-C transmissive materials includes materials that have greater than 10% transmission. In embodiments, the materials have 10% transmission. In embodiments, the materials have 11% transmission, 15% transmission, 20% transmission, 25% transmission, 30% transmission, 35% transmission, 40% transmission, 45% transmission, or 50% transmission. In embodiments, the UV-C transmissive material does not include material that transmits less than 1% of the light.

[0022] In other embodiments, the method provides that the UV-C light removes pathogens, microorganisms, cells, or cellular debris from the surface of the cell culture device. In some embodiments, the pathogens include bacteria and / or viruses.

[0023] In other aspects, methods for removing cells from a preselected region of a cell culture device are provided, and include seeding the cell culture device with mammalian cells, the mammalian cells growing within the cell culture device, and exposing a preselected region of the cell culture device to UV-C light at an amount and for a period of time effective to kill, inactivate or detach a microorganism in the preselected region, and where the microorganism includes pathogens, cells, viruses, or cellular debris.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] These and other features will be more readily understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0025] FIG. 1 is a perspective view of one embodiment of a cell culture device, the cell culture device is in the left panel and a chamber of the device is shown in the right panel;

[0026] FIG. 2 is a side view of the cell culture device of FIG. 1, showing the device being exposed to UV-C light to remove cells from the injection port; the collagen chamber 22 is depicted;

[0027] FIG. 3 is a side view of the cell culture device of FIG. 1, showing the UV-C treatment to expose and remove cells surrounding a 3D collagen chamber, with a blocking mask used to protect certain areas of the device from the UV-C light;

[0028] FIG. 4 is a side view of the cell culture device of FIG. 1, showing fluid flow used to carry away dead cells; where “X” refers to injection ports;

[0029] FIG. 5A is a photograph showing 10 minute exposure to UV-C with a coverslip; where the coverslip is a glass 170 pm coverslip;

[0030] FIG. 5B is a photograph showing 1 minute exposure with a cover slip;

[0031] FIG. 5C is a photograph showing 10 minute exposure with no cover slip;

[0032] FIG. 5D is a photograph showing 1 minute exposure with no coverslip;

[0033] FIG. 5E is a photograph showing UV-C treatment;

[0034] FIG. 6A is a photograph showing live / dead images of the cells using a BioRad Zoe at 1 minute exposure, with the coverslip blocking the UV-C exposure;

[0035] FIG. 6B is a photograph showing live / dead images of the cells using a BioRad Zoe at 1 minute exposure, with no coverslip;

[0036] FIG. 7A is a graph showing the % live cells verses relative UV-C exposure;

[0037] FIG. 7B is a graph showing the total cell count verses relative UV-C exposure;

[0038] FIG. 8 A is a graph showing the adsorption spectrum of PDMS;;

[0039] FIG. 8B is a graph showing the transmittance of glass; and

[0040] FIG. 9 is a photograph of a cell culture device with two injection ports, a 3D collagen chamber, and various channels for fluid flow.

[0041] It is noted that the drawings are not necessarily to scale. The drawings are intended to depict only typical aspects of the subject matter disclosed herein, and therefore should not be considered as limiting the scope of the disclosure.DETAILED DESCRIPTION

[0042] Certain illustrative embodiments will now be described to provide an overall understanding of the principles of the structure, function, manufacture, and use of the devices and methods disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will understand that the devices and methods specifically described herein and illustrated in the accompanying drawings are non-limiting illustrative embodiments and that the scope of the present invention is defined solely by the claims. The features illustrated or described in connection with one illustrative embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present invention.

[0043] Further, in the present disclosure, like-named components of the embodiments generally have similar features, and thus within a particular embodiment each feature of each like-named component is not necessarily fully elaborated upon. Additionally, to the extent that linear or circular dimensions are used in the description of the disclosed systems, devices, and methods, such dimensions are not intended to limit the types of shapes that can be used in conjunction with such systems, devices, and methods. A person skilled in the art will recognize that an equivalent to such linear and circular dimensions can easily be determined for any geometric shape.

[0044] Definitions

[0045] The following definitions are included for the purpose of understanding the present subject matter and for constructing the appended patent claims. The abbreviations used herein have their conventional meanings within the chemical and biological arts.

[0046] While various embodiments and aspects of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0047] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0048] The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.

[0049] In the descriptions herein and in the claims, phrases such as “at least one of’ or “one or more of’ may occur followed by a conjunctive list of elements or features. The term “and / or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and / or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and / or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.

[0050] It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “0.2-5 mg” is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.

[0051] As used in the description herein and throughout the claims that follow, the meaning of “a,” and “an” mean “at least one” and include plural reference unless the context clearly dictates otherwise. Likewise, the definite article “the” may include plural references.

[0052] Provided herein are methods and devices that enable use of spatial patterning of UV-C exposure to selectively remove cells from a cell culture device. Unwanted cells are removed from preselected regions of the cell culture device while leaving cells intact in desired regions. The methods and devices described herein provide advantages to current techniques. Forexample, existing designs of cell culture devices (e.g., chips) allow cells to grow within the injection port, thereby obscuring and complicating the desired cell culture environment, and not creating a true 3D tissue microenvironment. Additionally, prior techniques do not remove cells from the preselected areas of cell culture devices.

[0053] Thus, the methods and devices provided herein overcome the above obstacles and provide a means for selective removal of unwanted cells (or cellular debris or other pathogens) within a preselected region using UV-C light. For example, a preselected region (e.g., the injection port of a cell culture device) is exposed with UV-C light at a particular dosage / time, which then kills, inactivates and / or detaches the unwanted cells. Therefore, only the cells not exposed to the UV-C light will remain within the cell culture device. This creates the desired 3D tissue microenvironment within the cell culture device. The ability to remove cells selectively and with high precision within a cell culture device provides great advantages in preparing a cell culture device having a true 3D tissue microenvironment.

[0054] The materials of the cell culture device and / or the preselected region are UV-C transmissive, in that UV-C light can pass through. UV-C transmissive materials are described herein, and include silicone-based materials such as polydimethylsiloxane (PDMS). In other examples, fluid is passed through the cell culture device during the UV-C exposure to minimize the effect of the dead cells on the live cells. In other examples, fluid is passed through the cell culture device after the UV-C exposure to minimize the effect of the dead cells on the live cells. In embodiments, the effect of the dead cells on the live cells is apoptosis. Alternatively, regions where cells are desired to remain intact can be covered or masked with a material that is non- UV-C transmissive. As described in more detail below, multiple exposures of the UV-C light can be used to achieve a high removal of unwanted cells, for example where at least 90% of the undesired cells removed.

[0055] Therefore, the methods and devices are advantageous as they provide targeted removal (e.g., patterning) of cells using UV-C light. Also, as described in more detail below, the source of the UV-C light can be in the form of a UV-C laser or UV-C microscope (e.g., a microscope with epi illumination). In some examples, additional cell types can be seeded into voids as well. In embodiments, the void is the ability to seed another cell type in the void created by the laser that removed the cells. In some embodiments, the methods and devices herein provide an alternative (subtractive rather than additive method) to bioprinting. In embodiments, themethods and devices provided herein provide a mosaic of cells. In embodiments, the original cell tubule has cells removed in a pattern that allows the second cell type to fill in the holes created by the UV-C light (e.g., a laser). In embodiments, the original cell tubule has cells removed in a pattern that allows the second cell type to fill in the voids created by the UV-C light (e.g., the laser).

[0056] Cell Culture Device

[0057] Microfluidic devices for the generation of tissue-engineered microenvironments (TEM) have been developed. These devices contain a chamber filled with a three-dimensional matrix. The matrix contains tubular voids that can be populated with various cell types, resulting in tubular cell structures. These cell tubes are lumenally connected to fluidic channels of the devices and, thus, can be perfused with nutrient solutions, test substances, cell solutions or other fluids. Lumenal perfusion, and perfusion or diffusion through the matrix, allow for tight control of the micro-environmental conditions within the devices. Fluid pressure and shear stress are known to affect cell shape, proliferation, differentiation, and protein expression.

[0058] The fluidic devices may be designed as small chips made of poly dimethylsiloxane (PDMS) sandwiched between a glass plate and a polycarbonate plate. These tissue-engineered microenvironment chips (TEM-chips) are designed for generating in vitro models that reproduce the micro-architectural and functional parameters of various tissues and organs. Because the setup leads to tubular cell structures that are completely surrounded by matrix (for example gelled collagen I, fibrin, or combinations of collagen I, IV, and / or hyaluronan), direct contact of the cells with non-biological materials is prevented. Contact with tissue-derived proteins has been shown to support physiological behavior in vitro. On the other hand, contact with non- biological materials can adversely affect cellular responses.

[0059] The architecture of the TEM-chips allows for the generation of two or more tissue compartments that can be independently perfused and may be separated from one another by, for example, cellular barriers or other barriers. For example, a single tubular cell structure within a collagen matrix presents a two- compartment system, consisting of a lumenal compartment within the cell tube and an extralumenal compartment comprised by the surrounding matrix. Both compartments are separated by a layer of cells that form a barrier between “inside” and “outside”. This compartmentalized setup mimics the micro-architecture of many tissues andorgans, for example microvasculature, renal tubules, and seminiferous cell tubules. Importantly, this setup allows cells to polarize, which is especially important for tissues with barrier functions.

[0060] FIG. 1 illustrates one exemplary embodiment of a cell culture delivery device. As shown, the device 10 generally includes a cell injection port 12 having a bottom chamber 14, where cells are left after injection from the injection port 12. The bottom chamber is in fluid communication via a fluid delivery channel 16 to a collagen chamber 18.

[0061] UV-C Exposure Methods

[0062] In certain embodiments, cells can be seeded in the cell culture device (or a tissue- engineered microenvironment; TEM-chip) and allowed to grow and adhere. As shown in FIG. 1, the cells are injected through the injection port 12 on the device 10. The cells then grow within the cell culture device 10 to achieve a confluent and / or full-seeded device. After cell injection, residual cells are left at the bottom chamber 14 of the cell injection port 12. The cells can be removed from the preselected region using UV-C exposure.

[0063] In some embodiments the UV-C exposure occurs before seeding the cells into the cell culture device. In other examples, the UV-C exposure can be applied immediately after seeding the cells into the cell culture device. In other examples, the UV-C exposure is delayed anywhere from 1 hour to 2 days after seeding the device, or about 2 hours after seeding, or about 4 hours after seeding, or about 8 hours after seeding, or about 12 hours after seeding, or about 24 hours after seeding, or about 36 hours after seeding or about 24 hours after seeding. In other examples, and as described in more detail below, multiple UV-C exposures are applied, for example at regular, predetermined intervals. The exposure intensity (e.g., time of exposure) can vary as described herein. For example, the UV-C exposure includes a time period ranging from 1 second 2 minutes.

[0064] UV-C exposure can be applied to a preselected region (e.g., the cell injection port). As shown in FIG. 2, the UV-C exposure is applied using a lens 20 to focus the UV-C illumination to the preselected region. In another embodiment, collimated light and a mask (e.g., a blocking mask) is used to expose the UV-C light to the preselected region. As shown in FIG. 2, the cell culture device (chip) 10 is constructed with a UV-C transmissive bottom, and a mask 22 (e.g., a metal or other non-UV-C transmissive material) is applied to the area on the cell culture device where UV-C light is not desired. In particular, the mask 22 is applied to a region outside of the preselected region subjected to UV-C exposure, for example, the mask 22 is applied over acollagen chamber 18. A glass coverslip 24 is provided at the bottom of the injection port 14. In some embodiments, as shown in FIG. 3, cells in a preselected region 26 are exposed to the UV-C light, whereas the collagen chamber 18 has a mask 22 preventing exposure of UV-C light to the region.

[0065] The UV-C light, when applied, can be effective to kill, inactivate or detach cells from the preselected region. After the UV-C exposure to the preselected region, a fluid can be run through the cell culture device to remove the cells that were killed, inactivated or detached. In other examples, a fluid is run through the cell culture device during the UV-C exposure to the preselected region, which concurrently removes the killed, inactivated or detached cells. In other examples, a fluid is run through the cell culture device after the UV-C exposure to the preselected region, which concurrently removes the killed, inactivated or detached cells. The fluid that is run through the cell culture device during or after the UV-C exposure can include media, a buffer, saline, or other aqueous solution. The cells not exposed to the UV-C light (either that are not within the preselected region, or by being masked) remain within the cell culture device. For example, referring to FIG. 4, as cells are sufficiently exposed to the UV-C, they detach from the surface of the cell culture device, as indicated by the dots 28. Flow is administered, for example through an injection port 12 or other opening on the device 10, during or after exposure to remove the dead / dying and detached cells, while cells masked by the UV-C are retained, as shown by dots 30 within the collagen chamber 18. In other words, cells exposed to the UV-C light 28 (e.g., not covered by the mask 22) that are within the preselected region 26 detach and are washed away and removed from the device 10 by fluid.

[0066] UV-C Light

[0067] In some embodiments, the methods herein include exposing a preselected region of a cell culture device with UV-C light. In some examples, the UV-C light includes a laser, a light emitting diode (LED), a light box, a lamp, a laser, or a microscope objective.

[0068] In some embodiments, the UV-C light is applied (or exposed) to the preselected region at a wavelength between 200-300 nm. In certain embodiments, the wavelength is between 200-290 nm, or between 200-280 nm, or between 200-270 nm, or between 200-260 nm, or between 200- 250 nm, or between 200-240 nm, or between 200-230 nm, or between 200-220 nm, or between to 200-210 nm. In other embodiments, the wavelength is between 220-300 nm, or between 220- 290 nm, or between 220-270 nm, or between 220-260 nm, or between 220-250 nm, or between220-240 nm, or between 220-230 nm. In other embodiments the wavelength is between 250-300 nm, or between 250-290 nm, or between 250-280 nm, or between 250-270 nm, or between 250- 260 nm. In some examples, the wavelength is between 260-280 nm.

[0069] In some embodiments, the UV-C light is applied (or exposed) to the preselected region for a time period ranging from 1 second 2 minutes. In other examples, the exposure time of the UV-C light is about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 60 seconds, about 70 seconds, about 80 seconds, about 90 seconds, about 100 seconds, about 110 seconds, or about 120 seconds. In other embodiments the UV-C light is applied (or exposed) to the preselected region for over 2 minutes, for example about 3 minutes, about 4 minutes or about 5 minutes.

[0070] In other embodiments, the UV-C light is applied multiple times to the preselected region. For example, the UV-C light is applied 2 times, 3 times, 4 times, 5 times or more to the preselected region. In some embodiments, the UV-C light is applied multiple times at predetermined intervals. For example, the predetermined intervals can include regularly spaced time periods of every 2 hours, or every 6 hours, or every 12 hours, or every 24 hours or every 48 hours. In other embodiments, the UV-C light is applied at an irregular schedule, where for example, the intervals can include ramping up (e.g., every 2 hours for 8 hours), then ramping down (e.g., every 12 hours thereafter). In other embodiments, after the cells are seeded and grown within the cell culture device, the UV-C light is exposed to the preselected region and the unwanted cells are removed. After a period of time, the preselected region is re-exposed with the UV-C light to further eliminate unwanted cells.

[0071] In other embodiments, the UV-C light is applied to the preselected region after sufficient time for cells within the collagen tube (collagen chamber) to adhere. In some embodiments, this is measured by cell adhesion. For example, for mammalian cells, this can be from about 15 minutes to about 45 minutes, or 15 minutes, or 20 minutes, or 25 minutes, or 30 minutes, or 35 minutes, or 40 minutes or 45 minutes. In embodiments, cell adhesion is measured by the retention of the cells to the collagen tube surface. In embodiments, cell adhesion does not include cell clumping, where there is a significant amount of cell-cell adhesion. In embodiments, clumping refers to a number of cells not grown in a monolayer or not in a single monolayer.

[0072] In other examples, the UV-C light is applied after farming is completed and switching from growth media to sustaining media, one last UV-C treatment prior to shipping device.

[0073] UV-C transmissive materials

[0074] In certain embodiments, the cells (e.g., mammalian cells) can be seeded and grown within a cell culture device that is composed of a UV-C transmissive material. For example, UV-C transmissive refers to the ability of the material to transmit UV-C light, meaning that the UV-C light can pass through the particular material. As described above, UV-C light can include a wavelength between 200-300 nm. The UV-C transmissive material is capable of allowing the UV-C light to pass there through.

[0075] In some embodiments, the UV-C transmissive material includes polymeric organosilicon, silicone, polydimethyldiloxane (PDMS), polystyrene, polycarbonate, quarts TOP AS® 8007x1000, or cyclic olefin copolymer (COC). In some embodiments, the UV-C transmissive material is silicone or silicone based.

[0076] As described herein, glass transmits about 0.1% of the UV-C light, whereas silicone transmits about 30% of the UV-C light. In some embodiments, the UV-C transmissive material is a material that transmits at least about 15% of the UV-C light, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65% or about 70% or about 75% of the UV-C light. In embodiments, materials that do not transmit UV-C light include common plastics and common glasses. In embodiments, materials transmit more than 1% light below 300 nm.

[0077] In embodiments, the absorbance is proportional to optical depth, which includes the path length the light travels through the material. In embodiments, absorbance is dimensionless, and in particular is not a length, though it is an increasing function of path length, and approaches zero as the path length approaches zero.

[0078] In other examples, the UV-C transmissive material does not include glass, metal or other material that blocks the UV-C light.

[0079] Masking

[0080] As described above, the methods and devices can include masking applied to a particular region where UV-C light exposure is not desired (e.g., where the UV-C light is blocked). For example, in these regions, non-UV-C transmissive material may be applied. These materials include, for example glass, metal or other material that blocks the UV-C light. As describedherein, glass only transmits about 0.1% of the UV-C light, and thus can be used to mask UV-C light. In other examples, the material for masking can include a non-UV-C transmissive material with a defined thickness, e.g., a glass coverslip that is about 0.2 mm thick. In embodiments, the glass is from about 0.07 mm to 1.1 mm thick. In embodiments, the glass is 0.07 mm, or 0.08 mm, or 0.09 mm, or 0.10 mm, or 0.12 mm, 0.15 mm, or 0.18 mm, or 0.20 mm, or 0.25 mm, or 0.3 mm, or 0.35 mm, or 0.4 mm, or 0.45 mm, or 0.5 mm, or 0.55 mm, or 0.60 mm, or 0.65 mm, or 0.70 mm, or 0.75 mm, or 0.80 mm, or 0.85 mm, or 0.90 mm, or 1.0 mm, or 1.1 mm thick.

[0081] In some embodiments, the UV-C light is exposed to a preselected region on a cell culture device in a spatial pattern. For example, the spatial pattern can include a pulse that removes cells with a UV-blast. In other examples, the preselected region provides a mosaic pattern, wherein the UV-C light is transmitted in a mosaic pattern.

[0082] Pathogens and removal of pathogens

[0083] In some embodiments, cells (e.g., mammalian cells) are killed, inactivated or detached from a preselected region. In other embodiments, the UV-C light exposure removes pathogens, microorganisms, cells, and / or cellular debris from the preselected region. For example, the pathogen can include bacteria. In other examples, the pathogen can include viruses.

[0084] In some embodiments, after exposure of the UV-C light to the preselected region, at least about 70% of the mammalian cells are removed from the preselected region (e.g., by removed can include killed, inactivated and / or detached). In other embodiments, at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of mammalian cells are removed from the preselected region after UV-C exposure.

[0085] Shipping Device

[0086] In some embodiments, a cell culture device can be provided that is seeded with mammalian cells, allowing the cells to grow within the device (to generate a pre-seeded cell culture device). In some examples, the UV-C exposure can be applied prior to shipment of the pre-seeded cell culture device to a user. In other embodiments, the user can apply the UV-C light to the preselected region by any of the means described herein, for example using lensing, or collimated light, and / or masking.

[0087] Examples

[0088] Embodiments herein are further illustrated by the following examples and detailed protocols. However, the examples are merely intended to illustrate embodiments and are not to be construed to limit the scope herein.

[0089] Example 1 - Removal of cells with UV-C light

[0090] Cells were removed with a hand held UV-C light, and the data was repeated with a UV C light box. It was found that the glass cover slip blocked too much of the UV-C light (only about 0.1% of the UV-C light was transmitted). The system design has to permit the UV-C light to shine through, for example using a silicone chip material (e.g., PDMS). A 96 wells plate was used which showed a significant difference between treated and untreated.

[0091] Example 2 - UV-C treatment in-chip for elimination of cells

[0092] An exposure test was performed to evaluate the effectiveness of UV-C treatment to a preselected region of a cell culture device. hTERT-RPTEC cells were injected to the 96- well plate, and approximately 24,000 cells were seeded per well. Approximately 6-7 million cells were seeded to each well. The wells were exposed with the UV-C from the top of the plate and using aluminum foil to shield adjacent areas (e.g., mask the adjacent areas). The wells were exposed with a UV-C LED, specifically a Klaran LE-24V-3V-HC. Live / dead assays were performed. The cells were imaged, using a BioRad ZOE and Nikon (FIG. 5 and FIG. 6). As depicted in FIG. 5A, a 170 pm glass coverslip is provided showing that with a coverslip the cells still adhered to the device, and without a coverslip the cells are no longer attached, and are dying to be washed away from the device. As provided in FIG. 6A, the glass coverslip blocks exposure of the UV-C light. Without the coverslip, the cells are unattached and dead. In each of FIG. 6A and 6B, the same amount of cells were seeded.

[0093] The percent live cell count did not show a clear downward trend with increasing UV-C exposure and time. The total cell count (e.g., bound cells, or cells remaining after UV-C exposure) showed approximately a 90% drop with UV-C exposure at higher levels (with no coverslip); See Table 1 below and FIG. 7A and 7B. Unbound cells were removed during the washout for the live / dead assay.

[0094] The UV-C treated cells did not bind to the surface and were washed out.

[0095] Table 1 : Percent cell count

[0096] Chip material attenuation of UV-C at 260-270 nm was determined. Approximately 0.1% transmittance of 260-270 nm for glass coverslip was observed. Approximately 30% transmittance of 260-270 nm was observed through PDMS.

[0097] Lensing was used to focus UV-C light through PDMS at the cell injection port, e.g., from the top of the chip. An estimate of the relative difference between PDMS and glass (D263) transmission of 270 nm was determined (FIGS. 8A and 8B). In embodiments, the glass is a borosilicate glass. In embodiments, the glass is low fluorescent glass.

[0098] The cell count was determined in preselected areas of a cell culture device (see FIG. 9). Most cells were found in areas B (injection port 12), D (collagen chamber 18), and F (injection port 12). A previous measurement on Cytation CIO indicated that approximately 5,000 cells (hTERT cells) were in the cell tubule. It was determined that if cell injection ports (B (injection port 12) and F (injection port 12) of FIG. 9) and 125 pm channels (C (channel 16) and E (channel 16) of FIG. 9) were filled with cells, then approximately by calculation 18% of the cells were in the collagen tube (the 3D environment - “D” of FIG. 9), and assuming the same cell density everywhere. Approximately 25,000-50,000 were injected, see Table 2 below. Table 2 below provides the dimensions of the areas outlined in FIG. 9, along with the number of cells

[0099] Table 2: Cell count estimation (area letters reference to FIG. 9).

[0100] Table 3: Estimated cell count and cell density

[0101] It is estimated that the percentage of cells forming the tubule shifts from 18% to 57-69% given the effectiveness of UV-C treatment. The calculation assumes all cells affected by the UV-C treatment will wash out of the device (the chip).

[0102] In embodiments, the tubule or cell tubule refers to the cells grown in the chip with a cylindrical shape. The shape of the tubule is based on the mandrel the collagen is formed around. In embodiments, a “collagen tube” refers to the molded cylindrical void in the extracellular matrix (ECM) which does not have cells.

[0103] Additional methods are contemplated, which include UV-C treatment (UV-C exposure) shortly after cell seeding. For example, after sufficient time for cells in the collagen tube to adhere and flow unbound UV-C treated cells out of the chip. In other methods, it is contemplated that after farming is completed and switch from growth media to sustaining media, an additional UV-C treatment is performed prior to shipping the cell culture device.

[0104] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS:

1. A method, comprising: seeding a cell culture device with mammalian cells, the mammalian cells growing within the cell culture device, and exposing a preselected region of the cell culture device to ultraviolet-C (UV-C) light at an amount and for a period of time effective to kill, inactivate or detach the mammalian cells in the preselected region.

2. The method of claim 1, wherein the cell culture device comprises a chamber for growing the cells within an extracellular matrix (ECM).

3. The method of claim 1, wherein the cell culture device is configured to grow the cells in a cylindrical shape, within a cylindrical void in the extracellular matrix (ECM).

4. The method of claim 1, wherein the UV-C light is generated by a light source selected from the group consisting of a laser, a light emitting diode (LED), a light box, and a lamp.

5. The method of claim 1, wherein the UV-C light is focused through a lens.

6. The method of claim 1, wherein the preselected region comprises an injection port for delivering the mammalian cells to the cell culture device and wherein said preselected region comprises an area surrounding the injection port and / or an area outside the chamber for cell growth.

7. The method of claim 1, wherein the UV-C light is applied at a wavelength between OOSOO nm.

8. The method of claim 1 , wherein the preselected region is exposed to the UV-C light for a time period ranging from 1 second to 2 minutes.

9. The method of claim 1, wherein the UV-C light passes through at least a layer of a polymeric organosilicon compound, silicone, polydimethylsiloxane (PDMS), polystyrene, polycarbonate, quartz, Topas® 8007x100, a cyclic olefin copolymer, or other UV-C transmissive material.

10. The method of claim 9, wherein the silicone comprises polydimethylsiloxane (PDMS).

11. The method of claim 1 , wherein the preselected region is exposed multiple times with the UV-C light.

12. The method of claim 1, wherein the preselected region is exposed at an interval selected from the group consisting of every 2 hours, every 6 hours, every 12 hours, every 24 hours, and every 48 hours.

13. The method of claim 1, wherein at least about 70% or more of the mammalian cells are removed from the preselected region.

14. The method of claim 1, further comprising washing the killed, inactivated or detached mammalian cells from the cell culture device.

15. The method of claim 1, further comprising flowing a fluid through the cell culture device when exposing the preselected region to UV-C, or flowing a fluid through the cell culture device after exposing the preselected region to UV-C.

16. The method of claim 1, wherein the UV-C light is exposed in a spatial pattern to the preselected region.

17. The method of claim 16, wherein the spatial pattern comprises an area from about 1 mm2to about 5 mm2.

18. The method of claim 1, wherein at least a portion of the preselected region includes a mask that blocks the UV-C light.

19. The method of claim 18, wherein the mask comprises metal or a non-UV-C transmissive material.

20. The method of claim 1, wherein the cell culture device comprises UV-C transmissive material.

21. The method of claim 1, further comprising wherein the UV-C light removes pathogens, microorganisms, cells, or cellular debris from the surface.

22. The method of claim 21, wherein the pathogens comprise bacteria or viruses.

23. The method of claim 1, further comprising: exposing a second preselected region of the cell culture device to ultraviolet-C (UV-C) light at an amount and for a period of time effective to kill, inactivate or detach a microorganism in the preselected region, wherein the microorganism comprises pathogens, cells, viruses, or cellular debris.

24. A cell culture device for the generation of tissue-engineered microenvironments, the device comprising: a chip comprising a UV-C transmissible material interposed between two plates; at least one chamber within the UV-C transmissible material having a three-dimensional matrix therein, wherein the three-dimensional matrix surrounds a chamber adapted for cell growth; a source of UV-C light configured to selectively kill, inactivate and / or detach cells in a preselected region, in the chip.

25. The cell culture device according to claim 24, wherein the UV-C transmissible material is polydimethylsiloxane, and the two plates consist of a glass plate and polycarbonate plate.

26. The cell culture device according to claim 24, wherein the three-dimensional matrix comprises collagen, fibrin, hyaluronan, or combinations thereof.

27. The cell culture device according to claim 24, further comprising an injection port,28. The cell culture device according to claim 27, further comprising a mask preventing exposure of cells in the preselected region of the chip.

29. The cell culture device according to claim 28, wherein the mask is positioned to permit exposure to UV-C light of cells in a region around cell injection ports in the device and in the extracellular matrix outside the chamber in the device.

30. The cell culture device according to claim 24, wherein the source of UV-C light is a laser, a light emitting diode (LED), a light box, or a lamp.

31. The cell culture device according to claim 30, wherein the UV-C light is focused through a lens.

32. The cell culture device according to claim 24 wherein the chamber is tubular and adapted to grow tubular cellular structures therein.

33. The cell culture device according to claim 24, comprising tubular formations of mammalian cells in the chamber.

34. The cell culture device according to claim 33, wherein the tubular cell formations comprise a lumenal compartment within the tubular structure and an extraluminal compartment adapted to be separately perfused.

Images